The interaction of beta-arrestin with the AP-2 adaptor is required for theclustering of beta(2)-adrenergic receptor into clathrin-coated pits

beta-Arrestins are cytosolic proteins that regulate the signaling and the i nternalization of G protein-coupled receptors (GPCRs), Although termination of receptor coupling requires beta-arrestin binding to agonist-activated r eceptors, GPCR endocytosis involves the coordinate interactions between rec eptor-beta-arrestin complexes and other endocytic proteins such as adaptor protein 2 (AP-2) and clathrin, Clathrin interacts with a conserved motif in the beta-arrestin C-terminal tail; however, the specific molecular determi nants in beta-arrestin that bind AP-2 have not been identified. Moreover, t he respective contributions of the interactions of beta-arrestin with AP-2 and clathrin toward the targeting of GPCRs to clathrin coated vesicles have not been established. Here, we identify specific arginine residues (Arg(39 4) and Arg(396)) in the beta-arrestin 2 C terminus that mediate beta-arrest in binding to AP-2 and show, in vitro, that these domains in beta-arrestin 1 and 2 interact equally well with AP-2 independently of clathrin binding. We demonstrate in HEK 293 cells by fluorescence microscopy that beta(2)-adr energic receptor-beta-arrestin complexes lacking the beta-arrestin-clathrin binding motif are still targeted to clathrin-coated pits. In marked contra st, receptor-a-arrestin complexes lacking the beta-arrestin/AP-2 interactio ns are not effectively compartmentalized in punctated areas of the plasma m embrane. These results reveal that the binding of a receptor-beta-arrestin complex to AP-2, not to clathrin, is necessary for the initial targeting of beta(2)-adrenergic receptor to clathrin-coated pits.